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Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel.

Kienker PK, Wu Z, Finkelstein A - J. Gen. Physiol. (2015)

Bottom Line: Residues near either end of the TH6-TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6-TH7 segment resides at the cis interface.Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue's accessibility from either side.We also determined that this constriction is located near the middle of the TH8 helix.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, and Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 paul.kienker@einstein.yu.edu.

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Effect of trans MTS-ET on T-domain mutant L307C. The top trace is current and the bottom trace is voltage, with both plotted against time. After the addition of 4 ng L307C to the cis compartment (first arrow), the current at 30 mV gradually increased as channels formed in the membrane. Upon switching to negative voltage, the magnitude of the current rapidly decreased toward zero, indicating blocking by the amino-terminal His6-tag (Senzel et al., 1998); the current rapidly recovered upon returning to positive voltage. At the second arrow, after the channel activity had stabilized, 200 µg MTS-ET was added to the trans compartment, resulting in a decrease in current of ∼19%. The solutions were 1 M KCl, 2 mM CaCl2, 1 mM EDTA, with 20 mM malic acid, pH 5.3, in the cis compartment and 20 mM HEPES, pH 7.2, in the trans compartment. The break in the record was 3.6 min.
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fig2: Effect of trans MTS-ET on T-domain mutant L307C. The top trace is current and the bottom trace is voltage, with both plotted against time. After the addition of 4 ng L307C to the cis compartment (first arrow), the current at 30 mV gradually increased as channels formed in the membrane. Upon switching to negative voltage, the magnitude of the current rapidly decreased toward zero, indicating blocking by the amino-terminal His6-tag (Senzel et al., 1998); the current rapidly recovered upon returning to positive voltage. At the second arrow, after the channel activity had stabilized, 200 µg MTS-ET was added to the trans compartment, resulting in a decrease in current of ∼19%. The solutions were 1 M KCl, 2 mM CaCl2, 1 mM EDTA, with 20 mM malic acid, pH 5.3, in the cis compartment and 20 mM HEPES, pH 7.2, in the trans compartment. The break in the record was 3.6 min.

Mentions: We applied SCAM (Karlin and Akabas, 1998) to the TH6–TH7 segment in the hope of identifying channel-lining residues and perhaps also learning something about the secondary structure of this segment. We began by probing each mutant channel with the positively charged thiol-specific reagent MTS-ET. (MTS-ET had no effect on WT T-domain channels.) Fig. 2 shows a representative record of a macroscopic experiment, in this case with T-domain mutant L307C and trans MTS-ET. Note the characteristic His6-tag blocking at negative voltages and unblocking at positive voltage. We waited ∼10 min for the conductance at 30 mV to stabilize, and then added MTS-ET to the trans compartment. After a short delay because of the mixing time, the conductance dropped by a small amount, indicating reaction of trans MTS-ET with the cysteine residue. This is an indication that residue 307 is exposed in the channel. Table 1 summarizes the effects of cis and trans MTS-ET on the conductance of each mutant channel.


Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel.

Kienker PK, Wu Z, Finkelstein A - J. Gen. Physiol. (2015)

Effect of trans MTS-ET on T-domain mutant L307C. The top trace is current and the bottom trace is voltage, with both plotted against time. After the addition of 4 ng L307C to the cis compartment (first arrow), the current at 30 mV gradually increased as channels formed in the membrane. Upon switching to negative voltage, the magnitude of the current rapidly decreased toward zero, indicating blocking by the amino-terminal His6-tag (Senzel et al., 1998); the current rapidly recovered upon returning to positive voltage. At the second arrow, after the channel activity had stabilized, 200 µg MTS-ET was added to the trans compartment, resulting in a decrease in current of ∼19%. The solutions were 1 M KCl, 2 mM CaCl2, 1 mM EDTA, with 20 mM malic acid, pH 5.3, in the cis compartment and 20 mM HEPES, pH 7.2, in the trans compartment. The break in the record was 3.6 min.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4306713&req=5

fig2: Effect of trans MTS-ET on T-domain mutant L307C. The top trace is current and the bottom trace is voltage, with both plotted against time. After the addition of 4 ng L307C to the cis compartment (first arrow), the current at 30 mV gradually increased as channels formed in the membrane. Upon switching to negative voltage, the magnitude of the current rapidly decreased toward zero, indicating blocking by the amino-terminal His6-tag (Senzel et al., 1998); the current rapidly recovered upon returning to positive voltage. At the second arrow, after the channel activity had stabilized, 200 µg MTS-ET was added to the trans compartment, resulting in a decrease in current of ∼19%. The solutions were 1 M KCl, 2 mM CaCl2, 1 mM EDTA, with 20 mM malic acid, pH 5.3, in the cis compartment and 20 mM HEPES, pH 7.2, in the trans compartment. The break in the record was 3.6 min.
Mentions: We applied SCAM (Karlin and Akabas, 1998) to the TH6–TH7 segment in the hope of identifying channel-lining residues and perhaps also learning something about the secondary structure of this segment. We began by probing each mutant channel with the positively charged thiol-specific reagent MTS-ET. (MTS-ET had no effect on WT T-domain channels.) Fig. 2 shows a representative record of a macroscopic experiment, in this case with T-domain mutant L307C and trans MTS-ET. Note the characteristic His6-tag blocking at negative voltages and unblocking at positive voltage. We waited ∼10 min for the conductance at 30 mV to stabilize, and then added MTS-ET to the trans compartment. After a short delay because of the mixing time, the conductance dropped by a small amount, indicating reaction of trans MTS-ET with the cysteine residue. This is an indication that residue 307 is exposed in the channel. Table 1 summarizes the effects of cis and trans MTS-ET on the conductance of each mutant channel.

Bottom Line: Residues near either end of the TH6-TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6-TH7 segment resides at the cis interface.Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue's accessibility from either side.We also determined that this constriction is located near the middle of the TH8 helix.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, and Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 paul.kienker@einstein.yu.edu.

Show MeSH
Related in: MedlinePlus